Safe anaerobic metabolism: Naked mole-rats live in large colonies deep underground in hypoxic conditions. Park et al. found that these animals fuel anaerobic glycolysis with fructose by a rewired pathway that avoids tissue damage (see the Perspective by Storz and McClelland). These results provide insight into the adaptations that this strange social rodent has to make for life underground. They also have implications for medical practice, particularly for understanding how to protect tissues from hypoxia. Science , this issue p. 307 ; see also p. 248

As essential regulators of mitochondrial quality control, mitochondrial dynamics and mitophagy play key roles in maintenance of metabolic health and cellular homeostasis. Here we show that knockdown of the membrane-inserted scaffolding and structural protein caveolin-1 (Cav-1) and expression of tyrosine 14 phospho-defective Cav-1 mutant (Y14F), as opposed to phospho-mimicking Y14D, altered mitochondrial morphology, and increased mitochondrial matrix mixing, mitochondrial fusion and fission dynamics as well as mitophagy in MDA-MB-231 triple negative breast cancer cells. Further, we found that interaction of Cav-1 with mitochondrial fusion/fission machinery Mitofusin 2 (Mfn2) and Dynamin related protein 1 (Drp1) was enhanced by Y14D mutant indicating Cav-1 Y14 phosphorylation prevented Mfn2 and Drp1 translocation to mitochondria. Moreover, limiting mitochondrial recruitment of Mfn2 diminished formation of the PINK1/Mfn2/Parkin complex required for initiation of mitophagy resulting in accumulation of damaged mitochondria and ROS (mtROS). Thus, these studies indicate that phospho-Cav-1 may be an important switch mechanism in cancer cell survival which could lead to novel strategies for complementing cancer therapies.

Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or “spreading” of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.

Endothelial nitric oxide synthase (eNOS)–mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1–mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses, we observed increased interaction between eNOS and Cav-1 following stimulation of endothelial cells with thrombin, vascular endothelial growth factor, and Ca²⁺ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor l-N^G-nitroarginine methyl ester (hydrochloride) and Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine. We also observe increased binding of phosphomimicking Y14D-Cav-1 mutant transduced in human embryonic kidney cells overexpressing eNOS and reduced Ca²⁺-induced NO production compared to cells expressing the phosphodefective Y14F-Cav-1 mutant. Finally, Src FRET biosensor, eNOS small interfering RNA, and NO donor studies demonstrate NO-induced Src activation and Cav-1 phosphorylation at Tyr-14, resulting in increased eNOS/Cav-1 interaction and inhibition of eNOS activity. Taken together, these data suggest that activation of eNOS promotes Src-dependent Cav-1–Tyr-14 phosphorylation and eNOS/Cav-1 binding, that is, eNOS feedback inhibition.

We investigated the role of caveolae in the mechanism of increased pulmonary vascular permeability and edema formation induced by the activation of polymorphonuclear neutrophils (PMNs). We observed that the increase in lung vascular permeability induced by the activation of PMNs required caveolin-1, the caveolae scaffold protein. The permeability increase induced by PMN activation was blocked in caveolin-1 knockout mice and by suppressing caveolin-1 expression in rats. The response was also dependent on Src phosphorylation of caveolin-1 known to activate caveolae-mediated endocytosis in endothelial cells. To address the role of PMN interaction with endothelial cells, we used an intercellular adhesion molecule (ICAM)-1 blocking monoclonal antibody. Preventing the ICAM-1–mediated PMN binding to endothelial cells abrogated Src phosphorylation of caveolin-1, as well as the increase in endothelial permeability. Direct ICAM-1 activation by crosslinking recapitulated these responses, suggesting that ICAM-1 activates caveolin-1 signaling responsible for caveolae-mediated endothelial hyperpermeability. Our results provide support for the novel concept that a large component of pulmonary vascular hyperpermeability induced by activation of PMNs adherent to the vessel wall is dependent on signaling via caveolin-1 and increased caveolae-mediated transcytosis. Thus, it is important to consider the role of the transendothelial vesicular permeability pathway that contributes to edema formation in developing therapeutic interventions against PMN-mediated inflammatory diseases such as acute lung injury.